Many flavoprotein hydroxylase (external monooxygenases) are induced in soil microbes by and catalyze the hydroxylation of various stable organic compounds and toxicants produced industrially and naturally. The hydroxylated products are usually suitable for further catabolism, serving as carbon sources for microbes. Microbial hydroxylases are thus important in contributing to environment protection and detoxification. The long-term goals of the proposed program are to delineate how microbial hydroxylases function and how to regulate their activities, both in vitro and in vivo. Toward these goals, a basic understanding of the structural and catalytic properties of hydroxylases is of central importance to the development of the full potential of microbial hydroxylation actions for biosphere protection. The present proposal focuses on studies of the salicylate hydroxylase and luciferase-flavin reductase system. Specific objectives are along four lines: (1) Essential amino acid residues at active sites will be identified, by chemical modification, photoinactivation, and affinity labeling, and characterized. The distances between sites for substrates, prosthetic group, and specific probes will be determined by energy transfer. The modes of molecular motion of hydroxylase-bound pyridine nucleotide and flavin will be investigated by fluorescence spectroscopy. Special emphasis is placed upon the correlation of structural parameters with catalytic properties. (2) Flavoprotein hydroxylases exhibit both hydroxylase and oxidase activities. The mechanisms and regulations of such dual-activity properties will be investigated under both turnover and nonturnover conditions. Particularly, the functional role of flavin in such dual-activity catalysis will be examined. (3) Various flavins and low-temperature techniques will be applied to study the oxygenated enzyme-flavin intermediates which are active in both hydroxylase and oxidase activities. (4) Mechanism of Photobacterium fischeri flavin reductase will be elucidated by kinetic and chemical studies. The present proposal emphasizes structural and mechanistic studies of in vitro enzyme systems and, in the meantime, bears a long-term interest in the delineation of mechanism and regulation of hydroxylase actions in vivo.